Fluidic system for reagent delivery to a flow cell

ABSTRACT

A fluidic system that includes a reagent manifold comprising a plurality of channels configured for fluid communication between a reagent cartridge and an inlet of a flow cell; a plurality of reagent sippers extending downward from ports in the manifold, each of the reagent sippers configured to be placed into a reagent reservoir in a reagent cartridge so that liquid reagent can be drawn from the reagent reservoir into the sipper; at least one valve configured to mediate fluid communication between the reservoirs and the inlet of the flow cell. The reagent manifold can also include cache reservoirs for reagent re-use.

PRIORITY CLAIM

This application claims the benefit of, U.S. Provisional Application No.61/863,795, filed Aug. 8, 2013, currently pending, which is incorporatedherein by reference.

BACKGROUND

Embodiments of the present disclosure relate generally to apparatus andmethods for fluidic manipulation and optical detection of samples, forexample, in nucleic acid sequencing procedures.

Our genome provides a blue print for predicting many of our inherentpredispositions such as our preferences, talents, susceptibility todisease and responsiveness to therapeutic drugs. An individual humangenome contains a sequence of over 3 billion nucleotides. Differences injust a fraction of those nucleotides impart many of our uniquecharacteristics. The research community is making impressive strides inunraveling the features that make up the blue print and with that a morecomplete understanding of how the information in each blue print relatesto human health. However, our understanding is far from complete andthis is hindering movement of the information from research labs to theclinic where the hope is that one day each of us will have a copy of ourown personal genome so that we can sit down with our doctor to determineappropriate choices for a healthy lifestyle or a proper course oftreatment.

The current bottleneck is a matter of throughput and scale. Afundamental component of unraveling the blue print for any givenindividual is to determine the exact sequence of the 3 billionnucleotides in their genome. Techniques are available to do this, butthose techniques typically take many days and thousands upon thousandsof dollars to perform. Furthermore, clinical relevance of anyindividual's genomic sequence is a matter of comparing unique featuresof their genomic sequence (i.e. their genotype) to reference genomesthat are correlated with known characteristics (i.e. phenotypes). Theissue of scale and throughput becomes evident when one considers thatthe reference genomes are created based on correlations of genotype tophenotype that arise from research studies that typically use thousandsof individuals in order to be statistically valid. Thus, billions ofnucleotides can eventually be sequenced for thousands of individuals toidentify any clinically relevant genotype to phenotype correlation.Multiplied further by the number of diseases, drug responses, and otherclinically relevant characteristics, the need for very inexpensive andrapid sequencing technologies becomes ever more apparent.

What is needed is a reduction in the cost of sequencing that driveslarge genetic correlation studies carried out by research scientists andthat makes sequencing accessible in the clinical environment for thetreatment of individual patients making life changing decisions.Embodiments of the invention set forth herein satisfy this need andprovide other advantages as well.

BRIEF SUMMARY

The present disclosure provides a fluidic system that includes a reagentmanifold comprising a plurality of channels configured for fluidcommunication between a reagent cartridge and an inlet of a flow cell; aplurality of reagent sippers extending downward from ports in themanifold, each of the reagent sippers configured to be placed into areagent reservoir in a reagent cartridge so that liquid reagent can bedrawn from the reagent reservoir into the sipper; at least one valveconfigured to mediate fluid communication between the reservoirs and theinlet of the flow cell.

This disclosure further provides a reagent cartridge that includes aplurality of reagent reservoirs configured to simultaneously engage aplurality of reagent sippers of a fluidic system along a z dimensionsuch that liquid reagent can be drawn from the reagent reservoir intothe sippers, the reagent reservoirs arranged in x and y dimensions intotop, middle and bottom rows, wherein reagent reservoirs along top andbottom rows of the cartridge are deeper along the z dimension thanreagent reservoirs in one or more middle rows; and at least twointerface slots configured to engage with corresponding alignment pinsof the fluidic system.

Also provided is a multi-layer diffusion bonded reagent manifoldcomprising at least 10, 15, or at least 20 ports, each port configuredto pull reagent from a separate reagent reservoir via a sipper, whereinthe ports are in fluid communication with one or more channels of a flowcell via fluidic channels in the manifold.

This disclosure further provides a method of reagent re-use thatincludes a) drawing a liquid reagent from a reagent reservoir into acache reservoir, the cache reservoir in fluid communication with thereagent reservoir and at least one channel of a flow cell; b)transporting the reagent from the cache reservoir onto the at least onechannel of the flow cell; c) transporting at least 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100% of the reagent on the flow cell channel to thecache reservoir such that the liquid reagent from the flow cell is notdirected back to the reagent reservoir after contacting the flow cell;and d) repeating steps b) and c) to achieve re-use of the liquid reagenton the flow cell.

This disclosure further provides a sequencing method that includes thesteps of (a) providing a fluidic system comprising (i) a flow cellcomprising an optically transparent surface, (ii) a nucleic acid sample,(iii) a plurality of reagents for a sequencing reaction, and (iv) afluidic system for delivering the reagents to the flow cell; (b)providing a detection apparatus comprising (i) a plurality ofmicrofluorometers, wherein each of the microfluorometers comprises anobjective configured for wide-field image detection in an image plane inx and y dimensions, and (ii) a sample stage; and (c) carrying outfluidic operations of a nucleic acid sequencing procedure in thecartridge and detection operations of the nucleic acid sequencingprocedure in the detection apparatus, wherein (i) the reagents aredelivered to the flow cell by the fluidic system, (ii) wide-field imagesof the nucleic acid features are detected by the plurality ofmicrofluorometers, and (iii) at least some reagents are removed from theflow cell to a cache reservoir.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a fluidic system with reagent sippers interacting with areagent cartridge.

FIG. 1B shows an isometric view of a manifold assembly and displays anexample of a layout of fluidic channels within the manifold.

FIG. 2 shows a front perspective view of a manifold assembly havingreagent sippers, valves and alignment pints. It also shows sippers ofdifferent lengths.

FIG. 3 shows a top view of a manifold assembly displaying one possiblelayout of fluidic channels within the manifold.

FIG. 4 shows a cross section view of channels within a manifold,including a cross section views of a cache line, and a non-cache fluidicchannel.

FIG. 5 shows a variety of junctions for connecting a reagent port withtwo valves.

FIG. 6 shows a cross section view of a reagent cartridge having wells ofvarying depths.

FIG. 7A shows a simplified top view of cache lines in a manifoldassembly according to one embodiment.

FIG. 7B shows various stages of reagent re-use in a method that utilizesreciprocal flow of reagent from a cache line to a flow cell, followed bypartial refilling of the cache line from the flow cell.

FIG. 8 shows a top view of a reagent tray interface having reagent wellsand interface slots for alignment pins.

FIG. 9 shows a fluidics map for a fluidic system.

FIG. 10 shows a detailed view of reagent sippers including compliantsippers and a piercing sipper.

DETAILED DESCRIPTION

This disclosure provides fluidic systems and methods for providingreagents to a chamber such as a flow cell. A particularly usefulapplication is detection of an immobilized biological sample. Forexample, the methods and systems set forth herein can be used in nucleicacid sequencing applications. A variety of nucleic acid sequencingtechniques that utilize optically and non-optically detectable samplesand/or reagents can be used. These techniques are particularly wellsuited to the methods and apparatus of the present disclosure andtherefore highlight various advantages for particular embodiments of theinvention. Some of those advantages are set forth below for purposes ofillustration and, although nucleic acid sequencing applications areexemplified, the advantages can be extended to other applications aswell.

The fluidic systems set forth herein are particularly useful with any ofthe detection apparatus configurations and sequencing methods set forthin U.S. patent application Ser. No. 13/766,413 filed on Feb. 13, 2013and entitled “INTEGRATED OPTOELECTRONIC READ HEAD AND FLUIDIC CARTRIDGEUSEFUL FOR NUCLEIC ACID SEQUENCING,” the content of which isincorporated by reference in its entirety.

In particular embodiments, a sample that is to be detected can beprovided to a detection chamber using a fluidic system as providedherein. Taking the more specific example of a nucleic acid sequencingapplication, the fluidic system can include a manifold assembly that canbe placed into fluidic communication with one or more of reservoirs forholding sequencing reagents, reservoirs for holding sample preparationreagents, reservoirs for holding waste products generated duringsequencing, and/or pumps, valves and other components capable of movingfluids through a flow cell.

In particular embodiments a fluidic system can be configured to allowre-use of one or more reagents. For example, the fluidic system can beconfigured to deliver a reagent to a flow cell, then remove the reagentfrom the flow cell, and then re-introduce the reagent to the flow cell.An advantage of re-using reagents is to reduce waste volume and reducethe cost of processes that utilize expensive reagents and/or reagentsthat are delivered at high concentrations (or in high amounts). Reagentre-use takes advantage of the understanding that depeletion of reagentoccurs only or primarily at the flowcell surface, and therefore amajority of the reagent goes unused and may be subject to re-use.

FIG. 1A shows an exemplary fluidic system 100 having reagent sippers 103and 104 and valves 102 that exploits advantages of fluidic systems thatare provided by several embodiments set forth herein. The fluidic system100 includes a manifold assembly 101 that contains various fixedcomponents including, for example, reagent sippers, valves, channels,reservoirs and the like. A reagent cartridge 400 is present havingreagent reservoirs 401 and 402 configured to simultaneously engage a setof reagent sippers 103 and 104 along a dimension z such that liquidreagent can be drawn from the reagent reservoirs into the sippers.

Shown in FIG. 1B is an exemplary manifold assembly 101 that can be usedto provide liquid reagents from reagent reservoirs to a flow cell. Themanifold includes reagent sippers 103 and 104 extending downward in adimension z from ports in the manifold. The reagent sippers 103 and 104can be placed into one or more reagent reservoirs (not shown) in areagent cartridge. The manifold also includes channels 107 fluidlyconnecting the reagent sipper 103 to a valve 102 and valve 109. Thereagent sippers 103 and 104, the channels 107 and the valve 102 mediatefluid communication between the reagent reservoirs and a flow cell (notshown). Valves 102 and 109 may individually, or in conjunction, selectsippers 103 or 104, and through channels such as 107, mediate fluidcommunication between the reagent reservoirs and a flow cell (notshown).

The apparatuses shown in FIGS. 1A and 1B are exemplary. Furtherexemplary embodiments of the methods and apparatus of the presentdisclosure that can be used alternatively or additionally to the exampleof FIGS. 1A and 1B are set forth in further detail below.

FIG. 2 shows another exemplary manifold assembly having reagent sippersand valves. The manifold has alignment pins 105 protruding downward fromthe manifold in an axis parallel to the reagent sippers. The alignmentpins 105 are longer along the z dimension compared to the reagentsippers, although in alternative embodiments they can also be of equallength or shorter. The alignment pins 105 are configured to engage withone or more corresponding interface slots on a reagent cartridge (notshown). The reagent sippers 103 and 104 are coupled to the manifold viaports 106 that are housed in the manifold body. Reagent sippers 104 arelonger in comparison to reagent sippers 103, in order to draw liquidfrom reagent reservoirs of varying depth that corresponds to the depthof the reagent sipper 103 or 104. In alternative embodiments, sippers103 and 104 can be of equal lengths, or may switch dominant lengths.

Also shown in FIG. 2 are channels 107A and 107B which reside on separatex-y planes. Separate channels 107A and 107B can originate from a singlechannel which then bifurcates at a T-junction 109 generating multiplechannels residing on separate planes. The manifold directs liquidreagent from one sipper to one or more valves by having the channelswhich connect to a particular valve 102 reside either, entirely on thesame plane A, or a combination of plane A and B, while channels whichconnect to any other valve may share this characteristic of co-plane orinter-plane origination.

FIG. 3 shows a top view of a manifold assembly 101 displaying onepossible layout of fluidic channels within the manifold. Fluidicchannels 107A and 107B originate from a single port 106 and connect port106 to either valve 102A or 102B. Certain channels include a cachereservoir 108 which has sufficient volume to allow a quantity of liquidreagent to flow from a flow cell (not shown) to the cache reservoir 108such that liquid reagent from the flow cell is not directed back to thereagent reservoir (not shown) after contacting the flow cell. Also shownin FIG. 3 are exemplary positions of one or more alignment pin 105. Themanifold assembly shown in FIG. 3 also includes inlet ports 111 forshared buffers. Each of valves 102A and 102B are configured with inletports corresponding to each reagent port 106, and with a common outports 112 and 110 which fluidly connect to a flow cell and a waste port113 and 109 which fluidly connect to a waste receptacle.

As demonstrated by the exemplary embodiments above, a fluidic system fordelivering reagents from a reagent cartridge to a flow cell can includea reagent manifold comprising a plurality of channels configured forfluid communication between a reagent cartridge and an inlet of a flowcell. Use of a manifold in fluidic systems provides several advantagesover the use of tubing alone. For example, a manifold with fixedchannels reduces the likelihood of error during assembly, such asmisplacement of tubing attachments, as well as over- or under-tighteningof connections. In addition, a manifold provides ease of maintenance,allowing, for example, quick replacement of an entire unit rather thantime-intensive testing and replacement of individual lines.

The one or more of the channels of the manifold can include a fluidictrack through a solid material. The track can be of any diameter toallow desired level of fluid transfer through the track. The track canhave an inner diameter of, for example, less than 0.1 mm, 0.2 mm, 0.3 mm0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm,5 mm, 6 mm, 7 mm, 8 mm, 9 mm or less than 10 mm in diameter. The trackconfiguration can be, for example, straight or curved. Alternatively oradditionally, the track can have a combination of curved portions andstraight portions. The cross section of the track can be, for example,square, round, “D”-shaped, or any other shape that enables a desiredlevel of fluid transfer through the track. FIG. 4 exemplifies a fluidictrack through a manifold body and shows a cross section view of onetrack 302. The exemplary channel 302 shown in FIG. 4 has a “D” shapedcross section formed by a 0.65 mm diameter half circle fused with anadditional 0.65 mm×0.325 mm rectangle.

The channel between the sipper and the valve can be housed entirelywithin the manifold body. Alternatively or additionally, the channel caninclude one or more portions that are external to the manifold. Forexample, tubing such as, for example, flexible tubing can connect aportion of the fluidic track to another portion of the track on themanifold. Alternatively or additionally, flexible tubing can connect aflow cell to fixed fluidic components of the system, including, forexample, pumps, valves, sensors and gauges. As an example, flexibletubing can be sued to connect a flow cell or a channel of the presentsystem to a pump such as a syringe pump or a peristaltic pump.

The manifold body can be, for example, made of any suitable solidmaterial that is capable of supporting one or more channels therein.Thus, the manifold body can be a resin such as polycarbonate, polyvinylchloride, DELRIN® (Polyoxymethylene); HALAR®; PCTFE(PolyChloroTriFluoroEthylene); PEEK™ (Polyetheretherketone); PK(Polyketone); PERLAST®; Polyethylene; PPS (Polyphenylene Sulfide);Polypropylene; Polysulfone; FEP; PFA; High Purity PFA; RADEL® R; 316Stainless Steel; TEFZEL® ETFE (Ethylene Tetrafluoroethylene); TPX®(Polymethylpentene); Titanium; UHMWPE (Ultra High Molecular WeightPolyethylene); ULTEM® (polyetherimide); VESPEL® or any other suitablesolid material that is compatible with the solvents and fluidstransported through the channels of the manifold in the embodimentspresented herein. The manifold body can be formed from a single piece ofmaterial. Alternatively or additionally, the manifold body can be formedfrom multiple layers that are bonded together. Methods of bondinginclude, for example, the use of adhesives, gaskets, and diffusionbonding. The channels can be formed in the solid material by anysuitable method. For example, channels can be drilled, etched or milledinto the solid material. Channels can be formed in the solid materialprior to bonding multiple layers together. Alternatively oradditionally, channels can be formed after bonding layers together.

FIG. 5 shows a variety of junctions 300 for connecting a reagent port301 with two valves. In each example shown in FIG. 5, a port 301 isfluidly connected to a channel 302 which bifurcates into two channels302A and 302B with each channel supplying a different valve. In thefirst configuration, the junction splits fluid flow from port 301 tochannels 302A and 302B on separate layers of the manifold. In the secondand third configurations shown in FIG. 5, the junction 300 includes arounded square 303 split within a layer or a full round split 304 withina layer of the manifold.

The manifold assemblies presented here are configured for delivery ofliquid reagents from a reagent cartridge to a flow cell. Thus, themanifold can have any number of ports coupled to reagent sippers. Morespecifically, the number of ports can correspond to the number andconfiguration of reagent reservoirs in a reagent cartridge. In someembodiments, the manifold comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or at least 30 ports, each portconfigured to couple a reagent sipper to a channel in fluidcommunication with the at least one valve.

The fluidic systems presented herein can also include an array of sippertubes extending downward along the z dimension from ports in themanifold, each of the reagent sippers configured to be inserted into areagent reservoir in a reagent cartridge so that liquid reagent can bedrawn from the reagent reservoir into the sipper. The reagent sipperscan comprise, for example, a tubular body with a proximal end and adistal end. The distal end can taper to a sharp tip that is configuredto pierce a film or foil layer used as a seal over a reagent reservoirin a reagent cartridge. Various exemplary sipper tips are shown in FIG.10. The reagent sippers can be provided with, for example, a singlelumen running through the tubular body from the distal to the proximalend. The lumen can be configured to provide fluid communication betweenthe reagent cartridge on one end of the sipper and the reagent manifoldon the other end of the sipper. As shown in exemplary FIG. 2, reagentsippers 103 and 104 are coupled to the manifold via ports 106 that arehoused in the manifold body.

In some embodiments, as exemplified in FIG. 2, a subset of the reagentsippers is of a length that is shorter than other reagent sippers. Forexample, the length of the subset can be at least 1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, or at least 2.0 mm shorter than the other reagentsippers. The manifold and reagent sippers can be used in a device havingan elevator mechanism configured to move a reagent cartridgebi-directionally along the z dimension such that the reagent sippers areinserted into corresponding wells or reservoirs in the reagentcartridge. In certain embodiments, the reagent wells may be covered withprotective foils. Thus, an advantage of providing sippers of varyinglength is a reduction in the force required by the elevator mechanism toaccommodate a foil-piercing force when a reagent cartridge is broughtinto contact with the piercing sippers. The difference in sipper lengthcan advantageously correspond to the depth of reagent wells in a reagentcartridge, so that each sipper reaches a desired depth in itscorresponding reagent well when the sippers and the cartridge are in afully engaged position.

The sippers can be formed of any suitable material that allows fluidtransfer through a lumen and which is compatible with the solvents andfluids transported through the channels of the manifold in theembodiments presented herein. The sippers can be formed from a singletube. Alternatively or additionally, one or more sippers can be made ofmultiple segments that together form a sipper of a desired length anddiameter.

In some embodiments, at least one of the reagent sippers includes acompliant tip configured to flex when the tip impinges upon the bottomof a reagent well in a reagent cartridge. By flexing or deforming, acompliant tip allows the lumen of the sipper to more fully approach oreven contact the bottom of the reagent well, thereby reducing or eveneliminating the evacuation volume in the reagent well. A compliant tipcan be especially advantageous for uptake of sample or reagents wheresmall volumes are used, or in situations where it is desirable foruptake of most or all of the liquid in a reagent reservoir. The body ofthe sipper having a compliant tip can be made entirely of the sameflexible material as the tip. Alternatively or additionally, the body ofthe sipper can be made of a distinct material than the tip. Thecompliant tip can be made of any suitable material such that thecompliant tip may deform or yield when urged into contact with thebottom of a reagent reservoir. Some suitable materials include polymericand/or synthetic foams, rubber, silicone and/or elastomers, includingthermoplastic polymers such as polyurethane.

The fluidic systems presented herein may also include, for example,pumps and valves that are selectively operable for controlling fluidcommunication between the reservoirs and the inlet of the flow cell. Asexemplified by the manifold assembly shown in FIGS. 2 and 3, channeloutlets on the manifold can be configured to connect with correspondinginlet ports on the one or more valves such that each reagent channel isin fluid communication with an inlet port on the valve. Thus, via thereagent channels of the manifold, one or more or each of the inlet portscan be in fluid communication with a reagent sipper. Each of the one ormore valves can be configured with a common out port (110, 112) whichfluidly connects to an inlet of one or more lanes on a flow cell.Alternatively or additionally, each of the one or more valves can beconfigured with a waste port (109, 113) fluidly connected to one or morewaste receptacles.

In embodiments where the fluidic system comprises at least a first valveand a second valve, each valve can be configured to simultaneouslydeliver separate reagents across a first channel and a second channel ofa flow cell, respectively. Thus, one valve can deliver one reagent to afirst flow cell channel while the second valve can simultaneouslydeliver a different reagent to a second flow cell channel. As shown inexemplary embodiments of FIG. 9, valve A (VA) is fluidly connected toinlet V1 of the flow cell, which is a manifold to deliver reagents tolane 1 and lane 3. Similarly, valve B (VB) is fluidly connected to inletV2 situated on the opposite end of the flow cell, and which deliversreagents to lane 2 and lane 4. Inlets V1 and V2 are situated on oppositeends of the flow cell and the direction of reagent flow occurs inopposite directions for lanes 1 and 3 compared to lanes 2 and 4.

The fluidic systems described herein can be used advantageously forfluidic manipulation of flow cell channels during nucleic acidsequencing. More specifically, a fluidic system described herein can beoperably associated with a detection apparatus in a configuration fordetection of nucleic acid features in the flow cell by the detectionapparatus. In some embodiments, the detection apparatus can comprise aplurality of microfluorometers, wherein each of the microfluorometerscomprises an objective configured for wide-field image detection in animage plane in x and y dimension. The fluidic systems set forth hereinare particularly useful with any of the detection apparatusconfigurations set forth in U.S. patent application Ser. No. 13/766,413filed on Feb. 13, 2013 and entitled “INTEGRATED OPTOELECTRONIC READ HEADAND FLUIDIC CARTRIDGE USEFUL FOR NUCLEIC ACID SEQUENCING,” the contentof which is incorporated by reference in its entirety.

As an example, in particular nucleic acid sequencing embodiments, a flowcell that contains a plurality of channels can be fluidicallymanipulated and optically detected in a staggered fashion. Morespecifically, the fluidic manipulations can be carried out on a firstsubset of the channels in the flow cell while optical detection occursfor a second subset of the channels. For example, in one configurationat least four linear channels can be disposed parallel to each other inthe flow cell (e.g. channels 1 through 4 can be ordered in sequentialrows). Fluidic manipulations can be carried out on every other channel(e.g. channels 1 and 3) while detection occurs for the other channels(e.g. channels 2 and 4). This particular configuration can beaccommodated by using a read head having detectors positioned in aspaced apart configuration such that the objectives are directed toevery other channel of the flow cell. In this case, valves can beactuated to direct flow of reagents for a sequencing cycle toalternating channels while the channels that are being detected aremaintained in a detection state. In this example, a first set ofalternating channels can undergo fluidic steps of a first sequencingcycle and a second set of alternating channels undergo detection stepsof a second sequencing cycle. Once the fluidic steps of the first cycleare completed and detection steps of the second cycle are completed, theread head can be stepped over (e.g. along the x dimension) to the firstset of alternating channels and valves can be actuated to deliversequencing reagents to the second set of channels. Then detection stepsfor the first cycle can be completed (in the first set of channels) andfluidic steps for a third cycle can occur (in the second set ofchannels). The steps can be repeated in this way several times until adesired number of cycles have been performed or until the sequencingprocedure is complete.

An advantage of the staggered fluidic and detection steps set forthabove is to provide for a more rapid overall sequencing run. In theabove example, a more rapid sequencing run will result from thestaggered configuration (compared to fluidically manipulating allchannels in parallel followed by detection of all channels in parallel)if the time required for fluidic manipulation is about the same as thetime required for detection. Of course, in embodiments where the timingfor detection steps is not the same as the timing for fluidic steps, thestaggered configuration can be changed from every other channel to amore appropriate pattern to accommodate parallel scanning of a subset ofchannels while another subset of channels undergoes the fluidic steps.

An additional advantage to having fluid flow in opposite directions isto provide a means of comparison of individual microfluorometerperformance. For example, where multiple microfluorometers are used perflow cell lane, it can be difficult to distinguish if decreasedmicrofluorometer performance is caused by the detector or from decreasedchemistry efficiency from one end of the lane to the other. By havingopposing directions of liquid flow, microfluorometer performance acrossthe lanes can be compared, effectively distinguishing whether decreasedperformance is due to the microfluorometer or not.

A fluidic map for an exemplary fluidic system is shown in FIG. 9. Flowcell 2020 has four lanes each fluidically connected to one of twoindividual fluid lines FV and RV that are individually actuated by inletvalves VA and VB. Inlet valve VA and inlet valve VB control the flow offluid from sample reservoirs, SBS reagent reservoirs and amplificationreagent reservoirs in reagent cartridge or tray 2035 fluidicallyconnected to various ports within reagent manifold 2030.

Flow of fluids through the system of FIG. 9 is driven by two separatesyringe pumps 2041 and 2042. The syringe pumps are positioned to pullfluids through the fluidic system and each pump can be individuallyactuated by valves 2051 and 2052. Thus, flow though each channel of theflow cell can be individually controlled by a dedicated pressure source.Valves 2051 and 2052 are also configured to control flow of fluids towaste reservoir 2060.

FIG. 9 exemplifies a fluidic system in which fluids are pulled by theaction of downstream syringe pumps. It will be understood that a usefulfluidic system can use other types of devices instead of syringe pumpsto drive fluids including, for example, positive or negative pressure,peristaltic pump, diaphragm pump, piston pump, gear pump or Archimedesscrew. Furthermore, these and other devices can be configured to pullfluids from a downstream position with respect to a flow cell or to pushfluids from an upstream position.

FIG. 9 also exemplifies the use of two syringe pumps for four channelsof a flow cell. Thus, the fluidic system includes a number of pumps thatis less than to the number of channels in use. It will be understoodthat a fluidic system that is useful in a fluidic cartridge of thepresent disclosure can have any number of pumps, for example, anequivalent or fewer number of pumps (or other pressure sources) than thenumber of channels in use. For example, several channels can befluidically connected to a shared pump and a valve can be used toactuate fluid flow through an individual channel.

The fluidic system exemplified in FIG. 9 also includes a sensor BUB-4for detecting air bubbles, positioned along the fluid path RV betweenvalve VA and flow cell inlet V1. An additional air bubble sensor BUB-3is positioned along the fluid path FV between valve VB and flow cellinlet V2. It will be understood that a fluidic line that is useful in afluidic system of the present disclosure can include any number of airbubble sensors, pressure gauges, and the like. The sensors and/or gaugescan be located at any position along any part of the fluid path in thefluidic system. For example, a sensor or gauge can be positioned along afluidic line between one of the valves and the flow cell. Alternativelyor additionally, a sensor or gauge can be positioned along a fluidicline between a reagent reservoir and one of the valves, between a valveand a pump, or between a pump and an outlet or reservoir such as a wastereservoir.

A cross-section of an exemplary reagent cartridge is shown in FIG. 6.The reagent cartridge 400 shown in FIG. 6 includes wells 401 of varyingdepths along the z dimension compared to those of wells 402. Morespecifically, the reagent cartridge exemplified in FIG. 6 has wellsdesigned to accommodate the length of a corresponding reagent sipper(not shown) such that each sipper reaches a desired depth in itscorresponding reagent well when the sippers and the cartridge are in afully engaged position. In the reagent cartridge exemplified in FIG. 6,the wells are arranged in row or column along the y dimension, wherethose wells 401 on the outside of the row or column extend downwardfurther along the z dimension than those wells 402 on the inside of therow or column. Some or all of the wells can be of varying depths.Alternatively or additionally, some or all of the wells can be of thesame depth. When the sippers and the cartridge are in a fully engagedposition, the penetration depth of any sipper tip (i.e., the distancefrom the bottom surface of the well to the end of the sipper tip) can beequivalent to the penetration depth of any other sipper tip in any othergiven well in the reagent cartridge. The penetration depth of any sippertip need not be the same as the penetration depth of any other givenwell in the reagent cartridge. Where at least some reagent wells have adifferent well depth, the well depth can be, for example, at least 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, or at least 2.0 mm shorter than the other reagentsippers. Similarly, when the sippers and the cartridge are in a fullyengaged position, the penetration depth of any sipper tip can be atleast 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, or at least 2.0 mm different than thepenetration depth of any other sipper tip in the reagent cartridge.

A top view of an exemplary reagent tray interface having reagent wellsand interface slots for alignment pins is shown in FIG. 8. As shown inthe exemplary reagent cartridge 400 in FIG. 8, the cartridge includes aplurality of reagent reservoirs 401A, 401B, 402A and 402B. The reagentreservoirs in FIG. 8 are arranged in x and y dimensions into rows. Alsoshown in FIG. 8, the cartridge includes interface slots 403 and 404configured to engage with corresponding alignment pins of a manifoldassembly (not shown). The cartridge may also include protective foilcovering any number of the reagent wells or reservoirs, which can bepierced by piercing sippers when the cartridge is brought into contactwith the piercing sippers.

The reagent cartridges presented herein can include any number ofreagent reservoirs or wells. The reagent reservoirs or wells can bearranged in any format along the x and y dimensions to facilitatetransport and storage of reagents in the cartridge. Alternatively oradditionally, reagent reservoirs or wells can be arranged in any formatalong the x and y dimensions suitable for interaction with an array ofsipper tubes extending downward along the z dimension from ports in themanifold. More specifically, the reagent reservoirs or wells can bearranged in any format suitable for simultaneously engaging a matrix ofreagent sippers such that liquid reagent can be drawn from the reagentreservoir into the sippers.

Not all reagent wells need interact simultaneously with all sipper tubesof a manifold assembly. For example, the reagent cartridge can include asubset of one or more reagent reservoirs or wells that are configured toremain in a non-interacting state while other reservoirs or wells areengaged by an array of sipper tubes. As one example, a cartridgepresented herein can comprises a plurality of wash reservoirs arrangedin a configuration corresponding to the plurality of reagent reservoirs,whereby wash reservoirs are configured to simultaneously engage thereagent sippers when the reagent sippers are not engaged with thereagent reservoirs so that wash buffer can be drawn from the washreservoirs into the sippers. An exemplary embodiment is presented inFIG. 8, which shows a row of reagent wells 401A. The cartridge alsoincludes a row of corresponding wells 401B which retains the sameorientation in the x dimension with respect to each other, but which areoffset in they dimension from wells 401A. The offset wells 401B caninclude a wash buffer, for example, provided for rinsing sipper tubesand fluidic lines after using one cartridge and before using anothercartridge.

Alternatively or additionally, other reservoirs that are empty, or whichhold buffer, sample or other reagents can be present on the cartridge.The additional reservoirs can, but need not interact with a sipper tube.For example, a reservoir can be configured to be filled with waste oroverflow reagent or buffer over the course of cartridge use. Such areservoir may be accessed, for example via a port that does notinterface with a sipper tube.

To facilitate correct alignment of cartridge reservoirs withcorresponding sipper tubes, alignment slots can be positioned in thecartridge. For example, in particular embodiments where an array ofsipper tubes is removed from one set of reservoirs and translocated toanother set of reagent or wash reservoirs, alignment slots can bepositioned in the cartridge to ensure correct alignment of the array ofreagent sippers with one or both sets of reservoirs. As shown in FIG. 8,the exemplary cartridge includes alignment slots 404 which retain thesame orientation in the x dimension, but which are offset in theydimension with respect to corresponding alignment slot 403. A cartridgeof the embodiments presented herein can have any number of alignmentslots which provide suitable alignment with the features of a fluidicassembly. For example, a cartridge can include 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more alignment slots configured to engage with correspondingalignment pins of the fluidic system so that reagent sippers of thefluidic system are positioned in alignment with the reagent and/or washreservoirs.

In particular embodiments a fluidic system can be configured to allowre-use of one or more reagents. For example, the fluidic system can beconfigured to deliver a reagent to a flow cell, then remove the reagentfrom the flow cell, and then re-introduce the reagent to the flow cell.One configuration is exemplified in FIG. 7A, which shows a top view ofcache lines in a manifold assembly. As shown in the schematic in the topportion of FIG. 7A, a reagent cache can be used to maintain aconcentration gradient from most used to least used (fresh) reagent. Insome embodiments, the cache reservoir can be configured to reduce mixingof fluid within the cache reservoir, thereby maintaining a gradient ofliquid reagent along the length of the reservoir from the end proximalto the flow cell to the end distal to the flow cell. As reagent isdelivered back to the flow cell from the cache reservoir, the gradientis maintained such that reagent flowed across the flow cell forms agradient from most used to least used (fresh) reagent.

As exemplified the diagram in the bottom portion of FIG. 7A, manifoldfluidics can be configured such that a reagent reservoir is in fluidcommunication with the input port of a flow cell (not shown) via valveinlet 1804. Valve 1804 controls flow of fluids between flow cell (notshown) and each of CLM reservoir, SRE reservoir, IMF reservoir, and LAM1and LPM1 reservoirs. Channel 1802 fluidly connects CLM reservoir viaport 1801 with valve inlet 1804. A portion of channel 1802 includes areagent cache 1803 configured to hold a volume of reagent equivalent tothe volume of one or more lanes of flow cell (not shown). The increasedvolume of reagent cache 1803 compared with other portions of channel1802 allows used reagent to be stored for re-use while maintaining astock of unused reagent in the reagent reservoir, thereby avoidingcontaminating the unused reagent stock in the reagent reservoir withused reagent.

The configuration shown in FIG. 7A is exemplary. Other configurationsare possible as well to achieve re-use. For example, one or more of thecache reservoirs can have a volume that is 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%,750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or moreof the volume of a flow cell channel in fluid communication with thecache reservoir. Alternatively or additionally, the cache reservoir cancomprise sufficient volume to allow a quantity of liquid reagent in oneor more flow cell channels to flow to the cache reservoir such that theliquid reagent from the flow cell is not directed back to the reagentreservoir after contacting the flow cell. For example, the quantity ofliquid reagent can comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%,250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%,850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or more of theliquid reagent in one or more flow cell channels.

A cache reservoir as presented herein can be configured to reduce mixingof fluid within the cache reservoir. In some such embodiments, reducedmixing can thereby maintain a gradient of liquid reagent along thelength of the reservoir from the end proximal to the flow cell to theend distal to the flow cell. Alternatively or additionally, a cachereservoir as presented herein can comprise one or more mixing elementsconfigured to promote mixing of fluid within the cache reservoir. Anysuitable active or passive mixing element can be used in suchembodiments. For example, the mixing element could comprise baffleelements, curved structures or any other passive or active structural orfluidic feature configured to promote mixing as fluid is transportedacross a cache reservoir. Alternatively or additionally, any suitablepump, rotor, blade, inlet and the like can be used for active mixingwithin a cache reservoir.

A cache reservoir as presented herein can have any shape, volume andlength that is suitable for the purposes of a cache reservoir. Inspecific embodiments, cache reservoirs of any shape, volume and/orlength can be used in the fluidic systems presented herein which allow aquantity of liquid reagent in one or more flow cell channels to flow tothe cache reservoir such that the liquid reagent from the flow cell isnot directed back to the reagent reservoir after contacting the flowcell. For example, a cache reservoir can comprise a serpentine channel.By way of another example, a cache reservoir can comprise a channel ofcylindrical or non-cylindrical shape. Further, any number of fluidicchannels in the fluidic system presented herein can include one or moreindividual cache reservoirs.

A cache reservoir as presented herein can be in fluid communication witha pump configured to move liquid reagent from the cache reservoir to theflow cell and from the flow cell back to the cache reservoir, whereiningress of reagent to the flow cell and egress of reagent from the flowcell occur through the same port of the flow cell. Alternatively oradditionally, ingress of reagent to the flow cell and egress of reagentfrom the flow cell may occur through distinct ports of the flow cell andstill achieve reagent re-use. For example, the fluidic systems presentedherein can make use of any of the reuse reservoirs and configurationsdescribed in connection with the apparatus configurations set forth inU.S. patent application Ser. No. 13/766,413 filed on Feb. 13, 2013 andentitled “INTEGRATED OPTOELECTRONIC READ HEAD AND FLUIDIC CARTRIDGEUSEFUL FOR NUCLEIC ACID SEQUENCING,” the content of which isincorporated by reference in its entirety.

The schematic of FIG. 7B sets forth an exemplary illustration of are-use method presented herein that utilizes reciprocal flow of reagentfrom a cache line to a flow cell, followed by partial refilling of thecache line from the flow cell. In the state shown in the top panel ofFIG. 7B, cache reservoir 1903 containing 100 μL of reagent 1906 is influid communication with flow cell lanes 1905 via splitter 1904 andvalve 1911. Valve 1904 is actuated to allow reagent 1906 to flow to flowcell lanes 1905. At the same time, fresh reagent 1907 is pulled fromreagent reservoir to fill void left in cache reservoir 1903. After useof the reagent on the flow cell, valve 1911 directs a portion (75 μL) ofused reagent 1906 back into cache reservoir 1903. Another portion (25μL) of used reagent 1906 is diverted by valve 1911 to a wastereceptacle. At the end of cycle 1, cache reservoir 1903 has a gradientwith 25 μL fresh reagent 1907 and 75 μL used reagent 1906 across thelength of the cache reservoir. The cycle of reciprocal flow of reagentfrom cache reservoir to flow cell and back to cache reservoir isrepeated, with a portion (25 μL) of used reagent 1906 diverted at eachcycle by valve 1911 to a waste receptacle and the remainder of usedreagent 1906 is flowed back to cache reservoir 1903. At the end of foursuch repeated cycles, the cache reservoir 1903 contains 25 μL freshreagent 1910, 25 μL of reagent that has been used once 1909, 25 μL ofreagent that has been used twice 1908, and 25 μL of reagent that hasbeen used three times 1907.

The configurations shown in FIG. 7A and FIG. 7B are exemplary. Otherconfigurations are possible as well to achieve re-use of one or more ofthe reagents used in a particular process. It will be understood that insome reagent re-use configurations, fluidic configurations for reagentre-use will only be used for a subset of the reagents used in aparticular process. For example, a first subset of the reagents may berobust enough to be re-used whereas a second subset may be prone tocontamination, degradation or other unwanted effects after a single use.Accordingly, the fluidic system can be configured for re-use of thefirst subset of reagents, whereas the fluidics for the second set ofreagents will be configured for single use.

A particular reagent can be re-used any number of times desired to suita particular process. For example, one or more of the reagentsexemplified herein, described in a reference cited herein, or otherwiseknown for use in a process set forth herein can be re-used at least 2,3, 4, 5, 10, 25, 50 or more times. Indeed any of a variety of desiredregents can be re-used for at least as many times. Any portion of aparticular reagent can be diverted back to a cache reservoir for re-use.For example, one or more of the reagents exemplified herein, describedin a reference cited herein, or otherwise known for use in a process setforth herein can have 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% of the volume of reagent on one or more flow cell lanesdirected back to a cache reservoir for subsequent re-use. Alternativelyor additionally, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% of the volume of reagent on one or more flow cell lanes can bediverted to a waste receptacle or otherwise removed from subsequent useon a flow cell.

Fluidic configurations and methods for reagent re-use, althoughexemplified for a nucleic acid sequencing process, can be applied toother processes, in particular processes that involve repeated cycles ofreagent delivery. Exemplary processes include sequencing of polymerssuch as polypeptides, polysaccharides or synthetic polymers and alsoinclude synthesis of such polymers.

As demonstrated by the exemplary embodiments above, a method of reagentre-use can include steps of: a) drawing a liquid reagent from a reagentreservoir into a cache reservoir, the cache reservoir in fluidcommunication with the reagent reservoir and at least one channel of aflow cell; b) transporting the reagent from the cache reservoir onto theat least one channel of the flow cell; c) transporting at least 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% of the reagent on the flow cellchannel to the cache reservoir such that the liquid reagent from theflow cell is not directed back to the reagent reservoir after contactingthe flow cell; d) repeating steps b) and c) to achieve re-use of theliquid reagent on the flow cell. The one or more of the cache reservoirscan be in fluid communication with a pump configured to move liquidreagent from the cache reservoir to the flow cell and from the flow cellback to the cache reservoir, such that ingress of reagent to the flowcell and egress of reagent from the flow cell occur through the sameport of the flow cell. Alternatively or additionally, ingress of reagentto the flow cell and egress of reagent from the flow cell may occurthrough distinct ports of the flow cell and still achieve reagentre-use. In some embodiments, reagent from the flow cell that is nottransported to the cache reservoir in step c) can be diverted. As anexample, reagent from the flow cell that is not transported to the cachereservoir can be transported to a waste reservoir. Transport of reagentin one or both of steps b) and c) can be performed via a valve whichfluidly connects the cache reservoir and the flow cell. Transport ofreagent in one or both of steps b) and c) can be performed, for examplewith fluid flow in a single direction, or can be performed withreciprocating flow.

Embodiments of the present fluidic systems and methods find particularuse for nucleic acid sequencing techniques. For example,sequencing-by-synthesis (SBS) protocols are particularly applicable. InSBS, extension of a nucleic acid primer along a nucleic acid template ismonitored to determine the sequence of nucleotides in the template. Theunderlying chemical process can be polymerization (e.g. as catalyzed bya polymerase enzyme) or ligation (e.g. catalyzed by a ligase enzyme). Ina particular polymerase-based SBS embodiment, fluorescently labelednucleotides are added to a primer (thereby extending the primer) in atemplate dependent fashion such that detection of the order and type ofnucleotides added to the primer can be used to determine the sequence ofthe template. A plurality of different templates can be subjected to anSBS technique on a surface under conditions where events occurring fordifferent templates can be distinguished. For example, the templates canbe present on the surface of an array such that the different templatesare spatially distinguishable from each other. Typically the templatesoccur at features each having multiple copies of the same template(sometimes called “clusters” or “colonies”). However, it is alsopossible to perform SBS on arrays where each feature has a singletemplate molecule present, such that single template molecules areresolvable one from the other (sometimes called “single moleculearrays”).

Flow cells provide a convenient substrate for housing an array ofnucleic acids. Flow cells are convenient for sequencing techniquesbecause the techniques typically involve repeated delivery of reagentsin cycles. For example, to initiate a first SBS cycle, one or morelabeled nucleotides, DNA polymerase, etc., can be flowed into/through aflow cell that houses an array of nucleic acid templates. Those featureswhere primer extension causes a labeled nucleotide to be incorporatedcan be detected, for example, using methods or apparatus set forthherein. Optionally, the nucleotides can further include a reversibletermination property that terminates further primer extension once anucleotide has been added to a primer. For example, a nucleotide analoghaving a reversible terminator moiety can be added to a primer such thatsubsequent extension cannot occur until a deblocking agent is deliveredto remove the moiety. Thus, for embodiments that use reversibletermination a deblocking reagent can be delivered to the flow cell(before or after detection occurs). Washes can be carried out betweenthe various delivery steps. The cycle can then be repeated n times toextend the primer by n nucleotides, thereby detecting a sequence oflength n. Exemplary sequencing techniques are described, for example, inBentley et al., Nature 456:53-59 (2008), WO 04/018497; U.S. Pat. No.7,057,026; WO 91/06678; WO 07/123,744; U.S. Pat. No. 7,329,492; U.S.Pat. No. 7,211,414; U.S. Pat. No. 7,315,019; U.S. Pat. No. 7,405,281,and US 2008/0108082, each of which is incorporated herein by reference.

For the nucleotide delivery step of an SBS cycle, either a single typeof nucleotide can be delivered at a time, or multiple differentnucleotide types (e.g. A, C, T and G together) can be delivered. For anucleotide delivery configuration where only a single type of nucleotideis present at a time, the different nucleotides need not have distinctlabels since they can be distinguished based on temporal separationinherent in the individualized delivery. Accordingly, a sequencingmethod or apparatus can use single color detection. For example, amicrofluorometer or read head need only provide excitation at a singlewavelength or in a single range of wavelengths. Thus, a microfluorometeror read head need only have a single excitation source and multibandfiltration of excitation need not be necessary. For a nucleotidedelivery configuration where delivery results in multiple differentnucleotides being present in the flow cell at one time, features thatincorporate different nucleotide types can be distinguished based ondifferent fluorescent labels that are attached to respective nucleotidetypes in the mixture. For example, four different nucleotides can beused, each having one of four different fluorophores. In one embodimentthe four different fluorophores can be distinguished using excitation infour different regions of the spectrum. For example, a microfluorometeror read head can include four different excitation radiation sources.Alternatively a read head can include fewer than four differentexcitation radiation sources but can utilize optical filtration of theexcitation radiation from a single source to produce different ranges ofexcitation radiation at the flow cell.

In some embodiments, four different nucleotides can be detected in asample (e.g. array of nucleic acid features) using fewer than fourdifferent colors. As a first example, a pair of nucleotide types can bedetected at the same wavelength, but distinguished based on a differencein intensity for one member of the pair compared to the other, or basedon a change to one member of the pair (e.g. via chemical modification,photochemical modification or physical modification) that causesapparent signal to appear or disappear compared to the signal detectedfor the other member of the pair. As a second example, three of fourdifferent nucleotide types can be detectable under particular conditionswhile a fourth nucleotides type lacks a label that is detectable underthose conditions. In an SBS embodiment of the second example,incorporation of the first three nucleotide types into a nucleic acidcan be determined based on the presence of their respective signals, andincorporation of the fourth nucleotide type into the nucleic acid can bedetermined based on absence of any signal. As a third example, onenucleotide type can be detected in two different images or in twodifferent channels (e.g. a mix of two species having the same base butdifferent labels can be used, or a single species having two labels canbe used or a single species having a label that is detected in bothchannels can be used), whereas other nucleotide types are detected in nomore than one of the images or channels. In this third example,comparison of the two images or two channels serves to distinguish thedifferent nucleotide types.

The three exemplary configurations in the above paragraph are notmutually exclusive and can be used in various combinations. An exemplaryembodiment is an SBS method that uses reversibly blocked nucleotides(rbNTPs) having fluorescent labels. In this format, four differentnucleotide types can be delivered to an array of nucleic acid featuresthat are to be sequenced and due to the reversible blocking groups oneand only one incorporation event will occur at each feature. Thenucleotides delivered to the array in this example can include a firstnucleotide type that is detected in a first channel (e.g. rbATP having alabel that is detected in the first channel when excited by a firstexcitation wavelength), a second nucleotide type that is detected in asecond channel (e.g. rbCTP having a label that is detected in the secondchannel when excited by a second excitation wavelength), a thirdnucleotide type that is detected in both the first and the secondchannel (e.g. rbTTP having at least one label that is detected in bothchannels when excited by the first and/or second excitation wavelength)and a fourth nucleotide type that lacks a label that is detected ineither channel (e.g. rbGTP having no extrinsic label).

Once the four nucleotide types have been contacted with the array in theabove example, a detection procedure can be carried out, for example, tocapture two images of the array. The images can be obtained in separatechannels and can be obtained either simultaneously or sequentially. Afirst image obtained using the first excitation wavelength and emissionin the first channel will show features that incorporated the firstand/or third nucleotide type (e.g. A and/or T). A second image obtainedusing the second excitation wavelength and emission in the secondchannel will show features that incorporated the second and/or thirdnucleotide type (e.g. C and/or T). Unambiguous identification of thenucleotide type incorporated at each feature can be determined bycomparing the two images to arrive at the following: features that showup only in the first channel incorporated the first nucleotide type(e.g. A), features that show up only in the second channel incorporatedthe second nucleotide type (e.g. C), features that show up in bothchannel incorporated the third nucleotide type (e.g. T) and featuresthat don't show up in either channel incorporated the fourth nucleotidetype (e.g. G). Note that the location of the features that incorporatedG in this example can be determined from other cycles (where at leastone of the other three nucleotide types is incorporated). Exemplaryapparatus and methods for distinguishing four different nucleotidesusing detection of fewer than four colors are described for example inU.S. Pat. App. Ser. No. 61/538,294, which is incorporated herein byreference.

In some embodiments, nucleic acids can be attached to a surface andamplified prior to or during sequencing. For example, amplification canbe carried out using bridge amplification to form nucleic acid clusterson a surface. Useful bridge amplification methods are described, forexample, in U.S. Pat. No. 5,641,658; US 2002/0055100; U.S. Pat. No.7,115,400; US 2004/0096853; US 2004/0002090; US 2007/0128624; or US2008/0009420, each of which is incorporated herein by reference. Anotheruseful method for amplifying nucleic acids on a surface is rollingcircle amplification (RCA), for example, as described in Lizardi et al.,Nat. Genet. 19:225-232 (1998) and US 2007/0099208 A1, each of which isincorporated herein by reference. Emulsion PCR on beads can also beused, for example as described in Dressman et al., Proc. Natl. Acad.Sci. USA 100:8817-8822 (2003), WO 05/010145, US 2005/0130173 or US2005/0064460, each of which is incorporated herein by reference.

As set forth above, sequencing embodiments are an example of arepetitive process. The methods of the present disclosure are wellsuited to repetitive processes. Some embodiments are set forth below andelsewhere herein.

Accordingly, provided herein are sequencing methods that include (a)providing a fluidic system comprising (i) a flow cell comprising anoptically transparent surface, (ii) a nucleic acid sample, (iii) aplurality of reagents for a sequencing reaction, and (iv) a fluidicsystem for delivering the reagents to the flow cell; (b) providing adetection apparatus comprising (i) a plurality of microfluorometers,wherein each of the microfluorometers comprises an objective configuredfor wide-field image detection in an image plane in x and y dimensions,and (ii) a sample stage; and (c) carrying out fluidic operations of anucleic acid sequencing procedure in the cartridge and detectionoperations of the nucleic acid sequencing procedure in the detectionapparatus, wherein (i) the reagents are delivered to the flow cell bythe fluidic system, (ii) wide-field images of the nucleic acid featuresare detected by the plurality of microfluorometers, and (iii) at leastsome reagents are removed from the flow cell to a cache reservoir.

Throughout this application various publications, patents and/or patentapplications have been referenced. The disclosure of these publicationsin their entireties is hereby incorporated by reference in thisapplication.

The term comprising is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

What is claimed is:
 1. A fluidic system for delivering reagents from areagent cartridge to a flow cell comprising: a reagent manifoldcomprising a plurality of channels configured for fluid communicationbetween a reagent cartridge and an inlet of a flow cell; a plurality ofreagent sippers extending downward from ports in the manifold, each ofthe reagent sippers configured to be placed into a reagent reservoir ina reagent cartridge so that liquid reagent can be drawn from the reagentreservoir into the sipper; at least one valve configured to mediatefluid communication between the reservoirs and the inlet of the flowcell.
 2. The fluidic system of claim 1, wherein one or more of thechannels in the manifold comprise a cache reservoir.
 3. The fluidicsystem of claim 2, wherein one or more of the cache reservoirs has avolume that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least100% of the volume of a flow cell channel in fluid communication withthe cache reservoir.
 4. The fluidic system of claim 3, wherein the cachereservoir comprises sufficient volume to allow a quantity of liquidreagent in one or more flow cell channels to flow to the cache reservoirsuch that the liquid reagent from the flow cell is not directed back tothe reagent reservoir after contacting the flow cell.
 5. The fluidicsystem of claim 4, wherein the quantity of liquid reagent comprises 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% of the liquid reagent in one ormore flow cell channels.
 6. The fluidic system of claim 2, wherein oneor more of the cache reservoirs is in fluid communication with a pumpconfigured to move liquid reagent from the cache reservoir to the flowcell and from the flow cell back to the cache reservoir, wherein ingressof reagent to the flow cell and egress of reagent from the flow celloccur through the same port of the flow cell.
 7. The fluidic system ofclaim 6, wherein at least one valve is configured to differentiallydirect liquid reagent from the flow cell back to the cache reservoir orto the waste reservoir.
 8. The fluidic system of claim 2, wherein saidcache reservoir is configured to reduce mixing of fluid within the cachereservoir, thereby maintaining a gradient of liquid reagent along thelength of the reservoir from the end proximal to the flow cell to theend distal to the flow cell.
 9. The fluidic system of claim 2, whereinsaid cache reservoir comprises a plurality of mixing elements configuredto promote mixing of fluid within the cache reservoir.
 10. The fluidicsystem of claim 9, wherein the mixing elements comprise static featuresin the cache reservoir or on an interior surface of the cache reservoir.11. The fluidic system of claim 9, wherein the mixing elements comprisebaffle elements.
 12. The fluidic system of claim 2, wherein said cachereservoir comprises a serpentine channel.
 13. The fluidic system ofclaim 2, wherein said cache reservoir comprises a channel ofnon-cylindrical shape.
 14. The fluidic system of claim 1, wherein themanifold is configured to deliver reagent from a first reagent reservoirto a first valve via a first channel and from the first reagentreservoir to a second valve via a second channel.
 15. The fluidic systemof claim 14, wherein the manifold comprises a plurality of layers, andwherein each of the first and second channels resides in a separatelayer of the plurality of layers.
 16. A reagent cartridge comprising: aplurality of reagent reservoirs configured to simultaneously engage aplurality of reagent sippers of a fluidic system along a z dimensionsuch that liquid reagent can be drawn from the reagent reservoir intothe sippers, the reagent reservoirs arranged in x and y dimensions intotop, middle and bottom rows, wherein reagent reservoirs along top andbottom rows of the cartridge are deeper along the z dimension thanreagent reservoirs in one or more middle rows; and at least twointerface slots configured to engage with corresponding alignment pinsof the fluidic system.
 17. A multi-layer diffusion bonded reagentmanifold comprising at least 10, 15, or 20 ports, each port configuredto pull reagent from a separate reagent reservoir via a sipper, whereinthe ports are in fluid communication with one or more channels of a flowcell via fluidic channels in the manifold.
 18. A method of reagentre-use comprising: a) drawing a liquid reagent from a reagent reservoirinto a cache reservoir, the cache reservoir in fluid communication withthe reagent reservoir and at least one channel of a flow cell; b)transporting the reagent from the cache reservoir onto the at least onechannel of the flow cell; c) transporting at least 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100% of the reagent on the flow cell channel to thecache reservoir such that the liquid reagent from the flow cell is notdirected back to the reagent reservoir after contacting the flow cell;and d) repeating steps b) and c) to achieve re-use of the liquid reagenton the flow cell.
 19. A sequencing system comprising: a detectionapparatus; and a fluidic system as claimed in claim
 1. 20. A method ofsequencing nucleic acids, comprising: (a) providing a fluidic systemcomprising (i) a flow cell comprising an optically transparent surface,(ii) a nucleic acid sample, (iii) a plurality of reagents for asequencing reaction, and (iv) a fluidic system for delivering thereagents to the flow cell; (b) providing a detection apparatuscomprising (i) a plurality of microfluorometers, wherein each of themicrofluorometers comprises an objective configured for wide-field imagedetection in an image plane in x and y dimensions, and (ii) a samplestage; and (c) carrying out fluidic operations of a nucleic acidsequencing procedure in the cartridge and detection operations of thenucleic acid sequencing procedure in the detection apparatus, wherein(i) the reagents are delivered to the flow cell by the fluidic system,(ii) wide-field images of the nucleic acid features are detected by theplurality of microfluorometers, and (iii) at least some reagents areremoved from the flow cell to a cache reservoir.